A taq dna polymerase mutant with improved exonuclease activity and use thereof

Abstract

The application discloses a Taq DNA polymerase mutant with improved 5'→3' exonuclease activity and application thereof, and belongs to the technical field of enzyme engineering. The technical problem to be solved is that wild-type Taq DNA polymerase is insufficient in 5'→3' exonuclease activity in TaqMan probe qPCR, leading to low probe cutting efficiency, weak fluorescent signal, limited detection sensitivity (especially for low-abundance targets), slow reaction speed (high Ct value) and decreased specificity. The technical solution is to provide a Taq DNA polymerase mutant with high 5'→3' exonuclease activity, wherein the amino acid sequence of the Taq DNA polymerase mutant is shown as SEQ ID NO. 2. The mutant has 77% improved exonuclease activity, greatly accelerates the cutting speed of the TaqMan probe, accelerates the accumulation of the fluorescent signal, reduces the Ct value, and significantly shortens the detection time.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering technology, and relates to the directed evolution and modification of Taq DNA polymerase, specifically to a Taq DNA polymerase mutant with enhanced exonuclease activity and its applications. Background Technology

[0002] For understanding the technical content of this invention:

[0003] TaqMan probe-based real-time quantitative PCR (qPCR) is a quantitative analysis technique that uses real-time monitoring of PCR amplification products based on changes in fluorescence signals. Its core principle utilizes specific fluorescent probes, each labeled with a reporter fluorescent group and a quencher fluorescent group at both ends. During PCR amplification, the 5'-3' exonuclease activity of Taq DNA polymerase hydrolyzes the probe, separating the reporter and quencher fluorescent groups, thus generating a fluorescence signal. This technique offers advantages such as high specificity, accurate quantification, short experimental time, and compatibility with multiplex reactions, and is widely used in basic biological research, food testing, and medical diagnostics.

[0004] Wild-type Taq DNA polymerase has insufficient 5'→3' exonuclease activity in TaqMan probe-based qPCR. This deficiency leads to low probe cleavage efficiency, weak fluorescence signal, limited detection sensitivity (especially for low-abundance targets), slow reaction rate (high Ct value), high detection limit, decreased specificity, and susceptibility to interference from inhibitors. Summary of the Invention

[0005] The purpose of this invention is to provide:

[0006] A Taq DNA polymerase mutant with enhanced exonuclease activity and its application, as well as related technologies, to solve technical problems such as insufficient 5'→3' exonuclease activity of wild-type Taq DNA polymerase in TaqMan probe-based qPCR, or a combination thereof.

[0007] Terminology Explanation:

[0008] Unless otherwise defined, all technical terms in this document have the same meanings as commonly understood by one of ordinary skill in the art to which the subject matter of the claims pertains. Unless otherwise stated, all patents, patent inventions, and publications cited in this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms in this document, the definitions in this chapter shall prevail.

[0009] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0010] The definition of standard chemical terminology can be found in the reference "Molecular Biology, Science Press, by Huang Lihua".

[0011] Unless otherwise stated, conventional methods within the scope of the art, such as plasmid construction, plasmid transformation, cell culture, and sequencing, shall be used.

[0012] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0013] The term “Taq DNA polymerase (Taq DNAP)” used in this article refers to one of the most commonly used thermostable DNA polymerases in PCR technology.

[0014] The term “TaqMan probe-based qPCR” as used in this article refers to a real-time quantitative PCR technique that involves adding specific probes with fluorescent groups (such as FAM) and quenching groups (such as BHQ1) to the reaction system. During PCR extension, the 5'→3' exonuclease activity of Taq DNA polymerase cleaves the probes, releasing the fluorescent groups and generating a fluorescence signal. The fluorescence intensity is then used to quantify the target nucleic acid.

[0015] The term "5'→3' exonuclease activity" used in this article refers to the enzymatic ability of a nuclease to cleave phosphodiester bonds from the 5' end to the 3' end of a nucleic acid chain, releasing mononucleotides or oligonucleotides. In this invention, this activity is quantified by the "slope of the fluorescence value-time curve" (the higher the slope, the stronger the activity).

[0016] The term "Ct value" used in this article refers to the number of cycles required for the fluorescence signal to reach a set threshold in a qPCR reaction. A smaller Ct value indicates a higher initial concentration of the target nucleic acid and a stronger enzyme amplification / cleavage efficiency.

[0017] The term "specific activity" used in this article refers to the enzyme activity per unit mass of enzyme protein, expressed in "U / μg". "U" is defined as the amount of enzyme required to catalyze the incorporation of 10 nmold NTP into the DNA strand at 72°C for 30 min (refer to the product standard of Yisheng Bio 10101ES).

[0018] The term "DEPC water" as used in this article refers to ultrapure water (Grade I water) that has been treated with diethyl pyrocarbonate (DEPC) and sterilized under high temperature and high pressure. It is a colorless liquid and has been tested to be free of impurities such as RNA, DNA and protein.

[0019] In a first aspect, the present invention provides: a Taq DNA polymerase mutant, which is a mutation generated based on wild-type Taq DNA polymerase.

[0020] Among them, the technical feature mutation is selected from at least one of L30P, E507K and K508R.

[0021] Among them, the preferred technical feature mutation is a combination of L30P, E507K and K508R.

[0022] The sequence of the wild-type Taq DNA polymerase, which is a technical feature, is shown in SEQ ID NO.1.

[0023] SEQ ID NO.1:

[0024] MRGMLPLFEPKGRVLLVDGHHLAYRTFHALKGLTTSRGEPVQAVYGFAKSLLKALKEDGDAVIVVFDAKAPSFRHEAYGGYKAGRAPTPEDFPRQLALIKELVDLLGLARLEVPGYEADDVLASLAKKAEKEGYEVRILTADKDLYQLLSDRIHVLHPEGYLITPAWLWEKYGLRPDQWADYRALTGDESDNLPGVKGIGEKTARKLLEEWGSLEALLKNLDRLKPAIREKILAHMDDLKLSWDLAKVRTDLPLEVDFAKRREPDRERLRAFLERLEFGSLLHEFGLLESPKALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDLKEARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERLFANLWGRLEGEERLLWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEEIARLEAEVFRLAGHPFNLNSRDQLERVLFDELGLPAIGKTEKTGKRSTSAAVLEALREAHPIVEKILQYRELTKLKSTYIDPLPDLIHPRTGRLHTRFNQTATATGRLSSSDPNLQNIPVRTPLGQRIRRAFIAEEGWLLVALDYSQIELRVLAHLSGDENLIRVFQEGRDIHTETASWMFGVPREAVDPLMRRAAKTINFGVLYGMSAHRLSQELAIPYEEAQAFIERYFQSFPKVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVREAAERMAFNMPVQGTAADLMKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGEDWLSAKE。

[0025] Among them, the sequence of the technical feature Taq DNA polymerase mutant is the amino acid sequence shown in SEQ ID NO.2 or homologous to the amino acid sequence shown in SEQ ID NO.2 with a homology exceeding 99%, 99.4%, 99.7%.

[0026] SEQ ID NO.2:

[0027] .

[0028] Secondly, the present invention provides a nucleic acid that encodes any of the aforementioned Taq DNA polymerase mutants.

[0029] Thirdly, the present invention provides an expression vector that expresses any of the above-mentioned Taq DNA polymerase mutants or contains the above-mentioned nucleic acids.

[0030] Among them, the technical feature expression vector is a eukaryotic expression vector or a prokaryotic expression vector.

[0031] Fourthly, the present invention provides a cell comprising, expressing or secreting the above-described Taq DNA polymerase mutant; or comprising the above-described nucleic acid; or comprising the above-described expression vector.

[0032] Fifthly, the present invention provides the application of the above-mentioned Taq DNA polymerase mutant in TaqMan probe-based qPCR.

[0033] In a sixth aspect, the present invention provides products comprising the above-mentioned Taq DNA polymerase mutant, said products including kits and chips.

[0034] The technical features are used in the TaqMan probe-based qPCR reaction system.

[0035] In the reaction system, the volume ratio of TaqMan probe to Taq DNA polymerase mutant is 1-2:1-2.

[0036] The reaction conditions for the reaction system are: treatment at 90-100℃ for 1-3 minutes.

[0037] The present invention has at least the following beneficial effects:

[0038] The mutant in vitro cleavage activity was increased by 77%, which greatly accelerated the cleavage speed of the TaqMan probe, resulting in faster accumulation of fluorescence signal, a lower Ct value, and a significant reduction in detection time. Attached Figure Description

[0039] Figure 1 Fermentation of Taq DNA polymerase wild-type and mutant strains. -IPTG represents cells before the addition of inducer, +IPTG represents cells after induction; S represents the supernatant sample from ultrasonic disruption and centrifugation, P represents the precipitate sample from ultrasonic disruption; M represents protein markers, from top to bottom: 180kDa, 140kDa, 100kDa, 75kDa, 60kDa, 45kDa, 35kDa, 25kDa, 15kDa, 10kDa.

[0040] Figure 2 Purification of Taq DNA polymerase wild-type and mutant proteins. FT is the flow-through sample for affinity column loading; W is the sample retained after the washing process; 10%B, 50%B, and 100%B are elution samples with different concentrations of component B, respectively; M is the protein marker, with values ​​from top to bottom of 180kDa, 140kDa, 100kDa, 75kDa, 60kDa, 45kDa, 35kDa, 25kDa, 15kDa, and 10kDa.

[0041] Figure 3 This study describes the in vitro exonuclease activity assay for wild-type and mutant Taq DNA polymerase. The formula on the right shows the relationship between fluorescence value and reaction time for each experimental group, with the slope indicating the relative magnitude of exonuclease activity for each enzyme. Two independent replicates were performed for both mutant and wild-type assays.

[0042] Figure 4 This study compares the exonuclease activities of wild-type and mutant Taq DNA polymerases. The relative exonuclease activities measured in each group were averaged, and the relative differences were compared.

[0043] Figure 5 To detect residual Taq DNA polymerase after purification of wild-type and mutant samples. Based on the measured polymerization activities of wild-type and mutant samples, 10U samples were taken to test the levels of residual endogenous nickase, exonuclease, and RNase in the purified protein samples.

[0044] Figure 6 To compare the results of Taq DNA polymerase wild-type and mutant dye-based qPCR, 10U of enzyme was used as a template to react with cDNA formed by reverse transcription of total RNA from HEK293 cells in vitro.

[0045] Figure 7 for Figure 6 The melting curve of the corresponding dye-based qPCR is shown, and the afterimage is the original image exported from the device.

[0046] Figure 8 To compare the qPCR results of wild-type and mutant Taq DNA polymerase using the TaqMan probe method, 10U of enzyme was used for each reaction, with the HEK293 genome as a template.

[0047] Figure 9 for Figure 8 The melting curves for the corresponding TaqMan probe-based qPCR are shown, with the afterimages being the original images exported from the device. Detailed Implementation

[0048] Unless otherwise specified, all raw materials and reagents used in this invention were purchased from commercial suppliers, and experiments were conducted in accordance with the operating instructions. Unless otherwise specified, all instruments, equipment, and apparatus used in this invention are conventional instruments, equipment, and apparatus, and experiments were conducted in accordance with the operating instructions and the accompanying reagents.

[0049] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified in the embodiments, conditions are performed under conventional conditions or conditions recommended by the manufacturer. All reagents or instruments without specified manufacturers are commercially available conventional products. Numerous specific details are provided in the following detailed embodiments to better illustrate the invention. The specific embodiments described herein are for illustrative purposes only and are not intended to constitute any limitation on the invention.

[0050] Example 1

[0051] 1. Add 0.1mM MnCl2 to the reaction system of Yisheng Bio 10101ES product for PCR reaction. After collecting the product, construct the plasmid for recombination according to the Yisheng Bio 10911ES product manual.

[0052] 2. Transform the recombinant product into Rosetta (DE3) competent cells. Divide the recovered bacterial culture into 5 portions and spread them on solid LB plates containing carbenicillin antibiotic at a final concentration of 50 μg / mL. Incubate in an incubator at 37°C overnight (about 16 h) with the plates inverted.

[0053] 3. The following day, the number of single clones was calculated to be approximately 3.4 × 10⁻⁶. 3 Take 35 96-well deep-well plates and add 1.5 mL of fresh LB medium containing carbenicillin antibiotic to each well. Try to transfer clones that do not overlap on each plate and are of normal morphology and size into the well plate.

[0054] 4. After culturing in a shaker at 37°C and 800 rpm for about 2.5 h, remove the well plate and add IPTG to the well plate at a final concentration of 0.5 mM to induce protein expression. Incubate at 37°C and 800 rpm for 4-5 h.

[0055] 5. After preparing 100 μL of bacterial culture for each well, place each well in a centrifuge and centrifuge at 5000 rpm for 10 min at 4 °C, then discard the supernatant. Add 500 μL of 1×Taq buffer to each well, vortex to mix, centrifuge, and discard the supernatant. The 1×Taq buffer formula is as follows: 10 mM Tris-HCl, 50 mM KCl, 1.5 mM MgCl2, pH 8.9 at 25 °C;

[0056] 6. Add 500 μL of 1×Taq buffer to each well, shake to mix, and then transfer 300 μL of bacterial cells to a new 96-well plate. Sonicate the bacterial solution according to the recommended program of the ultrasonic disruptor, with parameters set as follows: power 60%, working for 4 seconds and stopping for 6 seconds, and ultrasonic disruption for 2 minutes.

[0057] 7. Centrifuge the well plate at 4°C and 5000 rpm for 10 min, and transfer 270 μL of the supernatant to a new 96-well plate.

[0058] 8. Set the water bath temperature to 85℃ in advance, seal the well plate and immerse it for 30 minutes to inactivate the extraneous proteins, then centrifuge to separate the supernatant;

[0059] 9. Calculate the required nucleic acid volume based on the amount of probe and substrate added to each well to a final concentration of 0.25 μM. Mix an appropriate amount of probe and substrate 1:1 under light-protected conditions and incubate at 95°C for 2 min, then immediately place on ice for later use. The probe and substrate concentration in the system is 1 μM. The probe and substrate sequences are as follows:

[0060] Probe (SEQ ID NO.3): 5'-FAM-GGTTGGTGTTGCGGAAGCCCAGGGT-BHQ1-3';

[0061] Substrate (SEQ ID NO. 4): 5'-ACCCTGGGCTTCCGCAACACCAACCACCTGATCAACAACAAGACCTGGAACATATGTTCCAGGTCTTGTTGTTGATCA-3'.

[0062] 10. Prepare the reaction system according to Table 1, aliquot and quickly place in a qPCR instrument. Detect the fluorescence value of the reaction wells every 30 seconds. Wild-type Taq DNA polymerase is placed in each of the four corners of each test plate as a control group for that plate.

[0063] Table 1

[0064]

[0065] 11. Record and collect the correlation between fluorescence values ​​and time for all samples, perform linear fitting, and compare the average slope of each well with that of the wild-type group on the same plate. Calculations showed that two mutant wells in this library had significantly higher slopes than the wild-type (1.5-fold), and sequencing confirmed that both corresponded to the amino acid sequences of SEQ ID NO.2. These mutants exhibited the following amino acid mutations compared to the wild-type: L30P / E507K / K508R.

[0066] Example 2

[0067] The following buffer solutions were used in the purification process:

[0068] Buffer A: 25mM Tris-HCl, 30mM NaCl, 0.5mM EDTA, 0.3% NP-40, 0.5% Tween-20, pH 8.0.

[0069] Buffer B: 25mM Tris-HCl, 1.0M NaCl, 0.5mM EDTA, 0.3% NP-40, 0.5% Tween-20, pH 8.0.

[0070] Buffer C: 50mM Tris-HCl, 200mM KCl, 0.5mM EDTA, 2.0mM DTT, 1.5% Tween-20, 1.5% NP-40, pH 8.0.

[0071] 1. After activating one clone, select a single clone and inoculate it into 4 mL of fresh LB medium. Add the appropriate amount of carbenicillin antibiotic and culture overnight.

[0072] 2. Inoculate the bacterial culture at a ratio of 1% into 800mL of fresh LB medium, add carbenicillin antibiotic to a final concentration of 50μg / mL, and incubate at 37℃ and 200rpm until the OD600 value is about 0.8. Then add 0.5mM IPTG and incubate overnight to induce protein expression.

[0073] 3. Collect the bacterial culture, centrifuge at 4℃ and 5000rpm for 30min and discard the supernatant. Resuspend the bacterial culture in 200mL of 1×PBS, centrifuge at 4℃ and 5000rpm for 30min and discard the supernatant.

[0074] 4. Weigh the total weight of the bacterial cells, resuspend the bacterial cells in 200mL of 1×PBS, and separate the resuspension corresponding to 5g of bacterial cells into a clean centrifuge tube.

[0075] 6. Balance the extracted suspension and the remaining suspension with 1×PBS, centrifuge at 4℃ and 5000rpm for 30min, and discard the supernatant. Use 5g of bacterial cells for purification, and store the remaining bacterial cells at -20℃ for later use.

[0076] 7. Resuspend 5g of bacterial cells in 50mL of buffer A, place them in an ultrasonic disruptor, and sonicate for 20min at 20% power with 3s ON / 3s OFF.

[0077] 8. After sonication, centrifuge the sample at 4°C and 12000 rpm for 30 min. Collect the supernatant, resuspend the precipitate in an equal volume of 1×PBS, and take 15 μL of both the supernatant and the precipitate resuspended for protein electrophoresis. Figure 1 As shown, both mutant and wild-type Taq DNA polymerases were expressed normally, and most of the target protein was found in the supernatant, i.e., expressed in a soluble form.

[0078] 9. Place the sonicated supernatant in a water bath and incubate at 85°C for 60 minutes with stirring to remove endogenous proteins with poor thermal stability.

[0079] 10. Equilibrate the purification column packed with 5 mL of HE packing material with 5 column volumes of buffer A at a flow rate of approximately 5 mL / min.

[0080] 11. Place the purification apparatus in a 4℃ constant temperature cabinet, load samples at a rate of 2 mL / min, and collect the flow-through solution;

[0081] 12. Elute with 10 column volumes of buffer A, collect the eluent, at a flow rate of approximately 5 mL / min;

[0082] 13. Rinse with 10 column volumes of 10% buffer B to remove weakly binding proteins at a flow rate of approximately 5 mL / min;

[0083] 14. Elute with 5 column volumes of 50% buffer B, then thoroughly wash the affinity column with 100% buffer B, and collect the eluent separately;

[0084] 15. Wash the affinity column with 10 column volumes of 3M NaCl and pure water respectively, and finally store the column in 20% ethanol solution.

[0085] 16. Take 10 μL of the liquid collected from each purification step and perform protein electrophoresis, such as... Figure 2 As shown, both mutant and wild-type Taq DNA polymerases were eluted in 50% buffer B with a purity greater than 95%.

[0086] 17. Transfer the eluent to a dialysis bag, immerse it in buffer C, and dialyze overnight at 4°C in a thermostat.

[0087] 18. Drain the liquid from the dialysis bag, add about 50% glycerol, and store in a -20°C refrigerator for later use.

[0088] Example 3

[0089] 1. Take 50 μL of both mutant and wild-type Taq DNA polymerase solutions, and dilute them in a 2-fold gradient with buffer C. Refer to the enzyme activity definition of Yisheng Bio's 10101ES to test the polymerization activity. Simultaneously, determine the protein concentration according to the Yisheng Bio Bradford Protein Assay Kit (20202ES) manual. The test data are shown in Table 2. The specific activity of the mutant is approximately 80% higher than that of the wild type, which helps to improve PCR yield and enzyme amplification performance.

[0090] Table 2

[0091]

[0092] 2. Take approximately 10 U of Taq DNA polymerase and add it to the reaction materials step by step according to the exonuclease activity test method in Example 1. Detect the absorbance of the reaction system every 30 seconds and perform linear fitting on the collected fluorescence signal changes. For example... Figure 3 As shown, the slope of the fluorescence trend line for each reaction well represents the relative exonuclease activity of the enzyme. Each protein was repeated twice.

[0093] 3. Summarize the slopes for each Taq DNA polymerase, using the average value to represent the relative exonuclease activity. For example... Figure 4 As shown, the relative exonuclease activity of the wild-type enzyme was 413.85, while that of the mutant was 732.43. The mutant exhibits an approximately 77% increase in relative exonuclease activity, which may result in a faster and stronger fluorescence signal under the same conditions in TaqMan qPCR applications.

[0094] Example 4

[0095] 1. Take three portions of the 10U Taq DNA polymerase mutant and wild-type enzyme solution prepared in Example 2 respectively, and place them on ice for later use;

[0096] 2. Endogenous nicking enzyme residue detection aims to detect the presence of nicking enzymes in the working amount of enzyme solution that could affect the application, causing damage to the PCR template and impacting the amplification process. Approximately 50 ng of freshly extracted pUC19 plasmid was incubated with Taq DNA polymerase at room temperature for 2 hours, followed by electrophoresis detection. Residual nicking enzymes can convert supercoiled plasmids to an open circular conformation, thereby altering their migration rate on agarose gels. Figure 5 As shown in the electrophoresis diagram of endogenous nicking enzyme, the total amount of supercoiled plasmids after incubation with wild-type and mutant enzyme solutions was not significantly different from that of the control, indicating that the endogenous nicking enzyme residue was acceptable.

[0097] 3. Endogenous exonuclease detection is performed to prevent residual exonuclease from consuming template, primers, and products, thus reducing PCR performance. 100 ng of λ DNA digested with Hind III is incubated with the enzyme solution at room temperature for 2 hours before electrophoresis. Exonuclease residues can alter the distribution of bands on the substrate during electrophoresis. For example... Figure 5 As shown in the exonuclease electrophoresis diagram, there was no significant difference in the substrate electrophoresis band pattern between wild-type and mutant enzyme solutions after incubation compared to the control, indicating that the endogenous exonuclease residue was acceptable.

[0098] 4. Endogenous RNases may affect enzyme performance in RNA-based applications such as RT-qPCR. For detection, 500 ng of freshly prepared HEK293 total RNA was incubated with the enzyme solution at room temperature for 4 hours, followed by electrophoresis. Figure 5 As shown in the RNase electrophoresis diagram, the RNA banding pattern in the experimental group was not significantly different from that in the control group, indicating that the endogenous RNase residue was acceptable.

[0099] Example 5

[0100] 1. Freshly extracted HEK293 RNA was reverse transcribed using the Yisheng Bio 11156ES Reverse Transcription Kit. The product was prepared and collected according to the instruction manual.

[0101] 2. Configure the qPCR reaction system according to Table 3, and perform three replicates for each enzyme. The HEK293 cDNA was the product reconstituted after ethanol precipitation in step 1, without DNA digestion. The two primers are located on two adjacent exons of β2-microglobulin. The amplification product is approximately 2500 bp when using DNA as a template and approximately 300 bp when using cDNA as a template. The template source can be determined by controlling the extension time.

[0102] Table 3

[0103]

[0104] The primer sequences are as follows:

[0105] PExon1 (SEQ ID NO. 5): 5'-TGAGTATGCCTGCCGTGTG-3'.

[0106] PExon2 (SEQ ID NO. 6): 5'-TCTCTGCTCCACCTCTAAGT-3'.

[0107] 3. Place the reaction tube in the qPCR instrument and set the program as shown in Table 4:

[0108] Table 4

[0109]

[0110] 4. Acquire fluorescence signal information and melting curves. For example... Figure 6 As shown, the mutant Taq DNA polymerase reacts 1.0 degrees earlier than the wild-type, but the fluorescence intensity at the plateau phase is comparable. This indicates that the final yields of both are at the same level, but the mutant Taq DNA polymerase has higher amplification efficiency. Figures 7-8 As shown, the melting curve peaks are single and concentrated, indicating that the system has good specificity and the amplification curve can truly reflect the amplification performance of the enzyme.

[0111] Example 6

[0112] 1. Genomic DNA was extracted from HEK293 cells. A TaqMan probe protocol was designed using the GITR (glucocorticoid-induced tumor necrosis factor receptor) gene as a template. The primer and probe sequences used are as follows:

[0113] GITR-F (SEQ ID NO. 7): 5'-AGTTCGGGTTTCTCACTGT-3'.

[0114] GITR-R (SEQ ID NO. 8): 5'-CCTCAGCTGCCAGATGTG-3'.

[0115] Probe (SEQ ID NO. 9): 5'-FAM-TCCCTGGGAACAAGACCCACAACGCTG-BHQ1-3'.

[0116] 2. Prepare the qPCR reaction system according to the system in Table 5, with 3 copies of each enzyme:

[0117] Table 5

[0118]

[0119] 3. Place the reaction tube in the qPCR instrument and set the program as shown in Table 6:

[0120] Table 6

[0121]

[0122] Fluorescence signals were collected every 30 seconds during amplification.

[0123] 4. Record and analyze the fluorescence signal and melting curve. For example... Figures 8-9 As shown, the melting curves of the two enzymes are uniform, the Ct value of the mutant is smaller than that of the wild type, and the fluorescence value is stronger than that of the mutant. This indicates that the enhanced exonuclease activity is beneficial for improving the application of Taq DNA polymerase in the TaqMan method.

[0124] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A Taq DNA polymerase mutant with enhanced exonuclease activity, characterized in that, The amino acid sequence of the Taq DNA polymerase mutant is based on the wild-type Taq DNA polymerase with the following mutations: L30P, E507K, and K508R. The amino acid sequence of the wild-type Taq DNA polymerase is shown in SEQ ID NO.

1.

2. A nucleic acid, characterized in that, Encodes the Taq DNA polymerase mutant of claim 1.

3. An expression carrier, characterized in that, The expression vector expresses the Taq DNA polymerase mutant of claim 1 or contains the nucleic acid of claim 2.

4. A cell, characterized in that, The cell comprises the Taq DNA polymerase mutant of claim 1, the nucleic acid of claim 2, or the expression vector of claim 3.

5. The application of the Taq DNA polymerase mutant according to claim 1 in TaqMan probe qPCR.

6. A product characterized in that, The product includes the Taq DNA polymerase mutant of claim 1, and the product includes kits.

7. A TaqMan probe-based qPCR kit, characterized in that, The TaqMan probe-based qPCR kit includes the Taq DNA polymerase mutant as described in claim 1.

8. The TaqMan probe-based qPCR kit according to claim 7, characterized in that, It also includes reverse transcriptase, buffer solution, dNTPs, and Mg. 2＋ One or more of the following: TaqMan probes, primers, and standards.

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